Trypanosoma brucei is the causative agent of African trypanosomiasis in humans and nagana in cattle. The parasite has a highly polarized cell shape that is optimized for evading the host immune response in its mammalian hosts and for migration to the salivary glands of its insect vector. One prominent feature of this polarity is th trypanosome's single flagellum, which nucleates in the posterior of the cell body, then extends adhered along the cell surface towards the anterior in a helical pattern. The positioning of the flagellum is essential for proper motility and must be inherited during cell division. It has been proposed that two cytoskeletal structures, one inside the cell body and one at the tip of the new flagellum, are responsible for ensuring that the new flagellum is positioned correctly. The flagellar attachment zone junction (FAZJ) and flagella connector (FC) contact a preexisting copy of the replicating structure and use it as a template or guide. This process, known as structural inheritance, is an important for conveying positional information from mother cell to daughter cell in a variety of organisms. While the FC and FAZJ have been studied morphologically, no components of either structure have been identified, which has made it difficult to confirm their functions and how they perform them. We have recently shown that the polo-kinase homolog in trypanosomes (TbPLK) localizes to both of these structures as it transits from the posterior to the anterior of the cell during division. Inhibition of the kinase causes severe defects in flagellr positioning, including detached flagella. During the course of performing in vivo biotinylation screens (BioID) and phosphoproteomics to identify TbPLK substrates and binding partners, we discovered proteins that are components of the FC and FAZJ. In the proposed work, we will determine the localization of both proteins in its two life cycle stages using fluorescence and electron microscopy to fully describe their position throughout the cell cycle. We will also establish the consequence of depleting the proteins by RNAi and gene knockout strategies to determine if the functions of the FAZJ and FC are consistent with theory. We will conduct additional BioID screens using the FAZJ and FC proteins to generate proteomes of both structures. These proteomes will more fully describe the components of these structures, which will help us explain how they function and if they have conserved elements with organelles in other organisms. Perturbing the assembly of these structures is a viable therapeutic avenue because it should inhibit the correct positioning of the new flagellum. Once this positional information is lost it is likely that it cannot be reestablished, which will severely hamper the daughter cell's motility and its ability to divide. Considering the lack of novel therapeutics for treating trypanosomiasis, finding novel pathways to exploit for drug targeting is essential.